Biomolecules such as proteins, polynucleotides, polysaccharides and the like have increasingly been gaining commercial significance as medicaments, as diagnostic agents, as additives for foodstuffs, detergents and the like, as research reagents and for many other applications. The need for such biomolecules cannot generally be satisfied by isolating the molecules from natural sources—e.g. in the case of proteins—but require the use of biotechnological production methods.
The biotechnological production of proteins typically begins with isolating the DNA which codes for the desired protein and cloning it into a suitable expression vector. After transfection of the expression vector into suitable prokaryotic or eukaryotic expression cells and subsequent selection of transfected cells the latter are cultivated in fermenters and the desired protein is expressed. Then the cells or the cultured supernatant are harvested and the protein contained therein is worked up and purified.
In the case of eukaryotic expression systems, i.e. when using mammalian cell cultures such as CHO or NSO cells, in the last 15 years a one hundred-fold increase has been achieved in the concentration of the desired protein which can be achieved in the cell cultures or cell culture supernatants in the expression step. Over the same period the binding capacity of chromatography materials which are used during the subsequent purification of the proteins has only improved by a factor of 3. For this reason there is an urgent need for improved, optimised purification processes for biomolecules, particularly proteins, which can be carried out on a large industrial scale.
In the case of biopharmaceuticals, such as proteins used as medicaments, e.g. therapeutic antibodies, in addition to the product yield the removal of impurities is also of outstanding importance. A distinction can be drawn between process-dependent impurities and product-dependent impurities. The process-dependent impurities contain components of the host cells such as proteins and nucleic acids and come from the cell culture (such as media ingredients) or from the working up (such as salts or detached chromatography ligands). Product-dependent impurities are molecular variants of the product with differing properties. These include shortened forms such as precursors and hydrolytic breakdown products, but also modified forms produced for example by deamination, incorrect glycosylations or wrongly linked disulphide bridges. The product-dependent variants also include polymers and aggregates. The term contaminants is used to denote all other materials of a chemical, biochemical or microbiological nature which do not directly belong to the manufacturing process. Examples of contaminants include viruses which may undesirably occur in cell cultures.
Impurities and contaminants lead to safety concerns in the case of biopharmaceuticals. These are intensified if, as is very often the case in biopharmaceuticals, the therapeutic proteins are administered by injection or infusion directly into the bloodstream. Thus, host cell components may lead to allergic reactions or immunopathological effects. In addition, impurities may also lead to undesirable immunogenicity of the protein administered, i.e. they may trigger an undesirable immune response by the patient to the therapeutic agent, possibly to the point of life-threatening anaphylactic shock. Therefore, there is a need for suitable purification processes by means of which all undesirable substances can be depleted to an insignificant level.
On the other hand, economic aspects cannot be ignored in the case of biopharmaceuticals. Thus, the production and purification methods used must not jeopardise the economic viability of the biopharmaceutical product thus produced. In addition, the timescale within which a new purification process can be established plays an important role: Besides its influence on the costs, the process development must be in tune with the preclinical and clinical development of the drug. Thus, for example, some of the preclinical and all the clinical trials can only begin when sufficient quantities of the biopharmaceutical of sufficient purity are available.
The following standard process consisting of four basic steps may serve as a starting point for developing a purification process for an antibody which can be carried out on a large scale: In the first step the target protein is isolated, concentrated and stabilised (“capturing”). In the second step, viruses are eliminated, in the third step purification is carried out in which the majority of the impurities such as nucleic acids, other proteins and endotoxins are depleted. In the final step any remaining traces of impurities and contaminants are eliminated (“polishing”).
In addition to filtration and precipitation steps, (column) chromatographic methods are of central importance. Thus, the capturing frequently includes a step of purification by affinity chromatography. Accordingly, there are numerous known column chromatographic methods and chromatography materials which can be used with them. With an increasing number of alternatives, ever greater numbers of preliminary trials have to be carried out, however, in order to determine the optimum materials and methods in terms of the purification effects, yield, biological activity, time, costs, etc.
When establishing and optimising a purification process it must also be borne in mind that it has to be tailored very individually to the biochemical and biophysical properties of the particular molecule to be purified (target molecule, target protein) and to the conditions under which the biological starting material was obtained. The biological starting material from which the target material has to be isolated generally consists of a very complex mixture of substances. In order to isolate and concentrate the target molecule, its specific properties such as shape, size, solubility, surface charge, surface hydrophobicity and biospecific affinity for binding partners are exploited. For each new target molecule, and even for the same target molecule with a variation in one of the preceding steps (e.g. a change in the composition of the cultivation medium for fermentation), the process has to be newly adjusted as it is possible that the best possible results from the above point of view will no longer be achieved under the new conditions.
At the same time, the number of theoretically conceivable alternatives in the process is potentiated by the number of parameters listed above. In column chromatography, for example, the arrangement of the chromatography step in the process as a whole, the column material, the pH, the salt content and the nature of the various eluant buffers used, the protein concentration when charging the column and many other aspects have to be optimised. This makes it virtually impossible to develop an optimised column chromatography process on an industrial scale at reasonable cost and in a reasonable time frame. On the other hand the economic viability and also equipment related restrictions (such as the need to use as few different buffers as possible and the smallest possible amounts or volumes of buffer and chromatography materials, to keep the volumes of product-containing fractions as small as possible and the need to minimise the processing times and also the volumes of waste water) demand such optimisation for each individual step of the process.
Conventionally, this problem is approached by successively varying a limited number of process parameters in more or less systematically conducted preliminary trials on the “trial and error” principle and ending these trials as soon as a basically “functioning” process can be found. Thus there is virtually no or only very limited systematic optimisation of all the essential parameters of a process, possibly from a number of points of view, e.g. with respect to the depletion of impurities, high product yields with simultaneously small losses of biological activity and the like. Processes established in such a way are consequently generally less than ideal.
Alternatively, attempts have been made to carry out the above optimisations using small columns on a laboratory scale (e.g. small columns containing about 1 ml of chromatography material). However, it was found that only a limited number of parameters could be varied at reasonable cost as the charging, washing and elution steps took a great deal of time, even on this smaller scale.
Another approach to optimising processes on a miniaturised scale can be found in WO2004/028658. The binding of a biological sample to chromatography materials is tested in parallel batches on multi-well plates in a batch method. Admittedly this process allows optimum conditions for this binding step to be determined quickly and cost-effectively. However, it is not possible to tell whether, under the conditions thus specified, optimum results can still be achieved in the column chromatography methods which are necessarily used under industrial-scale conditions. Also, this optimisation of the process relates only to optimising the step of charging the chromatography material, i.e. only a small part of the process as a whole.
Therefore, there is still an urgent need for processes by which column chromatographic methods of purification of biomolecules can be established and optimised quickly and cheaply, while these processes must also deliver satisfactory results under the conditions of large scale industrial production and purification of the biomolecule. The aim of the invention is to provide such a process.